Dementia risk and thalamic nuclei volumetry in healthy midlife adults: the PREVENT Dementia study

Abstract A reduction in the volume of the thalamus and its nuclei has been reported in Alzheimer’s disease, mild cognitive impairment and asymptomatic individuals with risk factors for early-onset Alzheimer’s disease. Some studies have reported thalamic atrophy to occur prior to hippocampal atrophy, suggesting thalamic pathology may be an early sign of cognitive decline. We aimed to investigate volumetric differences in thalamic nuclei in middle-aged, cognitively unimpaired people with respect to dementia family history and apolipoprotein ε4 allele carriership and the relationship with cognition. Seven hundred participants aged 40–59 years were recruited into the PREVENT Dementia study. Individuals were stratified according to dementia risk (approximately half with and without parental dementia history). The subnuclei of the thalamus of 645 participants were segmented on T1-weighted 3 T MRI scans using FreeSurfer 7.1.0. Thalamic nuclei were grouped into six regions: (i) anterior, (ii) lateral, (iii) ventral, (iv) intralaminar, (v) medial and (vi) posterior. Cognitive performance was evaluated using the computerized assessment of the information-processing battery. Robust linear regression was used to analyse differences in thalamic nuclei volumes and their association with cognitive performance, with age, sex, total intracranial volume and years of education as covariates and false discovery rate correction for multiple comparisons. We did not find significant volumetric differences in the thalamus or its subregions, which survived false discovery rate correction, with respect to first-degree family history of dementia or apolipoprotein ε4 allele status. Greater age was associated with smaller volumes of thalamic subregions, except for the medial thalamus, but only in those without a dementia family history. A larger volume of the mediodorsal medial nucleus (Pfalse discovery rate = 0.019) was associated with a faster processing speed in those without a dementia family history. Larger volumes of the thalamus (P = 0.016) and posterior thalamus (Pfalse discovery rate = 0.022) were associated with significantly worse performance in the immediate recall test in apolipoprotein ε4 allele carriers. We did not find significant volumetric differences in thalamic subregions in relation to dementia risk but did identify an interaction between dementia family history and age. Larger medial thalamic nuclei may exert a protective effect on cognitive performance in individuals without a dementia family history but have little effect on those with a dementia family history. Larger volumes of posterior thalamic nuclei were associated with worse recall in apolipoprotein ε4 carriers. Our results could represent initial dysregulation in the disease process; further study is needed with functional imaging and longitudinal analysis.


Introduction
Alzheimer's disease is the most common cause of dementia, characterized by cognitive decline associated with the presence of amyloid beta plaques and neurofibrillary tangles. 1 Some of the earliest symptoms that present during the prodromal stages include disturbances in mood, sleep and motivation. 2 Cognitive domains affected include episodic memory, visuospatial awareness, executive function, attention, speech and language. 2nherited autosomal dominant genetic mutations in the amyloid precursor protein (APP) and presenilin 1 (PSEN1) and 2 (PSEN2) genes cause early-onset Alzheimer's disease, which presents before the age of 65 years. 3Mutations in these genes result in the excess production and accumulation of amyloid beta 42 (Aβ 42 ). 3Individuals with these mutations display a strong family history for early-onset Alzheimer's disease. 3Most cases of sporadic Alzheimer's disease are of late onset, typically presenting in individuals over 65 years of age and associated with several genetic risk factors, particularly possession of the apolipoprotein ϵ4 (APOE4) allele. 4Homozygote APOE4 carriers have a greater risk of developing Alzheimer's dementia than heterozygotes. 4][7][8] Much of the focus on brain changes at the earliest stages of disease have been on the hippocampus and medial temporal lobe. 93][14][15] Volume loss in the hippocampus and the thalamus appears to precede atrophy of other structures such as the amygdala, where a volume reduction is only seen in people with Alzheimer's dementia. 16he thalamus comprises of 25 nuclei in each brain hemisphere (Table 1). 17Multiple studies have shown changes in the thalamic nuclei in Alzheimer's dementia. 18,19At autopsy, amyloid plaques have been found in almost all thalamic nuclei in Alzheimer's dementia, especially the anteroventral nucleus. 20Neurofibrillary tangles have been identified in the anterior and intralaminar thalamic subregions, and the laterodorsal and anteroventral nuclei. 20][23] Only a few studies have investigated volumetric changes in the thalamic nuclei in individuals with early-and late-onset Alzheimer's disease and MCI.Asymptomatic and symptomatic PSEN1 and APP mutation carriers have been found to have reduced thalamic volumes compared to healthy controls, while those with dementia exhibited a reduction in temporal and parietal volumes. 24A reduction in the volumes of the mediodorsal, pulvinar and medial geniculate (MGN) nuclei has been identified in early MCI. 25 The same nuclei, with the addition of the anteroventral and centromedian nuclei, are significantly smaller in individuals with late MCI and Alzheimer's dementia compared to healthy controls. 25an de Mortel et al. 26 found the volume of the ventrolateral thalamus was reduced in those with early MCI compared to those with normal cognition.Hippocampal and temporal atrophy only became evident in late MCI and Alzheimer's dementia, suggesting that thalamic pathology may be one of the first signs of cognitive decline. 26Low et al. 27 have identified significantly smaller anterior and posterior thalamic subnuclei volumes and greater leftward ventral thalamic atrophy in Alzheimer's dementia compared to MCI and healthy controls.
The thalamus plays a key role in cognition, especially in recall and processing speed. 28,29Deficits in episodic memory and executive function have been noted, with executive dysfunction being linked to damage in the medial thalamus. 30,31ndividuals exhibit difficulties with cognitive flexibility following a thalamic stroke. 31They also display difficulties with recognition and a greater response time, which is associated with volume loss of the mediodorsal lateral nuclei. 32ther case reports of thalamic strokes involving the mediodorsal medial (MDm) and lateral territory have reported greater problems with recall rather than recognition. 33rocessing speed has been shown to be related to the thalamus in individuals with multiple sclerosis. 34,35Atrophy of the anterior and superior left thalamus has been associated with slower processing speeds. 29ollectively, there is evidence suggesting the thalamus is affected early in the pathological course of Alzheimer's disease and may even predate hippocampal changes. 26halamic pathology may be implicated in the initial cognitive changes during the prodromal stages of Alzheimer's disease.A better understanding of the sequence of structural brain changes and cognitive performance can identify biomarkers to prevent dementia in those at risk.
We aimed to investigate differences in the volumes of the thalamus and its nuclei and the effect on cognitive performance with respect to dementia family history (FHD) and APOE4 carriership in a middle-aged, cognitively normal cohort, without signs of global atrophy. 36We hypothesized the volume of the thalamus would be reduced in APOE4 carriers compared to non-carriers and also reduced in individuals with FHD compared to those without.We further hypothesized that volumetric loss would be more prominent in the anterior and medial thalamic nuclei and would be related to recall ability.

Participants
Seven hundred cognitively healthy (absence of dementia or other neurological or major psychiatric conditions) participants aged between 40 and 59 years were recruited to the PREVENT Dementia study, which includes four sites in the UK (Oxford, Cambridge, Edinburgh and West London) and one in Ireland (Dublin).The study protocol has been described extensively before. 37A variety of recruitment strategies were used with the aim of purposefully

Cognitive analysis
Participants underwent cognitive testing using the computerized assessment of information (COGNITO) battery. 38This computerized cognitive battery tests a wide range of cognitive domains, such as attention, language, visuospatial awareness and memory, and has demonstrated good testretest reliability. 38The following measures and tests were used for the cognitive analyses: (i) Number of names correctly recalled in the immediate recall test (test of recall ability).Participants were asked to list the nine names that had been read aloud to them immediately after acquisition.(ii) Number of names correctly recalled in the delayed recall test (test of recall ability).Participants had to recite the names they had previously learned after completing other tests.(iii) Number of elements correctly recalled in the descriptive recall test (test of recall ability).Subjects had to recall 27 visual descriptive words or phrases they had been told as part of a short descriptive excerpt.(iv) Number of elements correctly recalled in the narrative recall test (test of recall ability).Participants had to recall 27 elements that had been told to them in a logical sequence as part of a story.(v) Time to first click in the visual and auditory attention test (test of working memory), which was used as a marker of processing speed.Participants were shown a shape and had to click on the identical shape while counting the sounds that were being played.(vi) Number of correct trials in the Stroop test (test of cognitive flexibility).Participants had to match the name of a colour to the button containing the name of the colour, a picture of the colour to the name of the colour and the name of the colour written in that colour to the colour name button. ][34][35]39

Image analysis
As part of the PREVENT Dementia MRI protocol, a T 1 -weighted magnetization-prepared rapid gradient echo sequence was acquired on 3 T Siemens (Siemens Healthcare, Erlangen, Germany) scanners with models varying depending on the scanning site (Prisma, Verio, Prisma fit and Skyra).
Participants were excluded if they did not have an MRI scan (n = 55) available.The remaining 645 T 1 -weighted MRI scans were analysed using FreeSurfer 7.1.0software and in particular the recon-all pipeline.Subsequently, a thalamic nuclei segmentation module, which is part of the FreeSurfer software, was applied (https://freesurfer.net/ fswiki/ThalamicNuclei) to segment the thalamic nuclei.This software has been validated to segment thalamic nuclei and is based on a probabilistic map determined from postmortem histology samples and ex vivo MRI brain scans. 17his is a validated atlas and demonstrates good test-retest reliability. 17It can identify 25 nuclei in each thalamic hemisphere.These nuclei were grouped into the following regions: anterior, lateral, ventral, intralaminar, medial and posterior, as suggested by Iglesias et al. 17 and shown in Table 1.Following segmentation, all scans underwent quality control, which was based on visual inspection of the scans.Scans were excluded from analysis if they included incidental findings, pathological lesions, or if the segmentation process had failed.This involved scans where the segmentation was missing several contiguous voxels or contained a lesion, as seen in Fig. 1.Fifteen scans were excluded due to pathological findings, and a further 15 scans because of failed segmentation, leaving 615 included for further analysis.
Thalamic volumes underwent ComBat harmonization to account for differences in the scanning equipment across the five sites.ComBat harmonization is used to account for the variability attributed to different sites and/or scanners. 40Age, sex, years of education, APOE4 and FHD were used as modulating variables.Four subjects were excluded from analysis looking at the effect of FHD due to incomplete information about their FHD.Four different subjects had incomplete information about APOE4 status and were excluded for analyses involving APOE4 status.Six hundred and eleven participants were included for analyses involving FHD, and 611 subjects were included for analysis involving APOE4 status.

Statistical analysis
Statistical analyses were carried out using R Statistical Software (version 4.1.0).A Shapiro-Wilk test was used to assess normality.Robust linear regression was used with age, sex, years of education and total intracranial volume as covariates.Initial analyses were confined to the thalamus and its six subregions, as suggested by Iglesias et al., 17 to identify any subtle changes in this young cohort.A false discovery rate (FDR) method was applied to correct for multiple comparisons.A P < 0.05 was considered statistically significant.
For any regions with a significant P FDR -value, further analysis investigating the nuclei within that region, as shown in Table 1, was undertaken with FDR correction.Chi-squared and Wilcoxon rank-sum tests were used to check for any statistical differences in baseline characteristics between the two groups.Volumetric differences of the thalamus and its subregions were compared with respect to FHD and APOE4 status.An interaction analysis was carried out to evaluate the effect of volumes of the regions of interest (ROIs) and FHD or APOE4 on cognitive performance.The effect of interactions between age and FHD and age and APOE4 on volumes of the ROI was investigated.Spearman's correlation was used to determine the directionality of significant interactions.

Results
The baseline characteristics for those included are summarized in Table 2.There were significant differences in the number of APOE4 carriers between FHD-positive and FHD-negative groups (P = 0.002) and the mean age (P = 0.037) and number of individuals with FHD (P = 0.002) between APOE4 carriers and non-carriers.

Volumetric differences with respect to FHD
We did not find a significant difference in the volume of the whole thalamus when comparing FHD-positive and FHD-negative groups (t = −0.68,P = 0.497).However, individuals with a positive FHD had a trend towards a smaller medial thalamus compared to FHD-negative group (t = −2.417,P = 0.016, P FDR = 0.095, β = −0.080),as shown in Fig. 2.

Volumetric differences with respect to APOE4 status
We did not find a difference in the volume of the thalamus with respect to APOE4 status (t = 0.105, P = 0.917).We also did not find any volumetric differences of the thalamic subregions with respect to APOE4 that survived FDR correction.
FHD status alone had no impact on the latency of responses in the visual and auditory attention test, as a marker of processing speed.However, there was a significant interaction between FHD and volume of the medial thalamus (t = 2.659, P = 0.008, P FDR = 0.048) in predicting processing speed as displayed in Fig. 4 and Supplementary Fig. 2. Within the medial thalamus, there were significant interactions between the MDm nucleus and FHD (t = 2.831, P = 0.005, P FDR = 0.019) in predicting processing speed.A larger volume of the MDm in those without FHD was associated with shorter time to first click, indicating a faster processing speed (Supplementary Table 2; medial thalamus, ρ = −0.255,P < 0.001; MDm, ρ = −0.260,P < 0.001).We did not find any significant interactions between the volumes of the ROI and FHD status on performance on the immediate, delayed, descriptive and narrative recall tests and Stroop test.
We did not find any significant interactions between the volumes of the ROI and APOE4 status in predicting performance in the delayed, descriptive and narrative recall tests, processing speed or Stroop test.

Discussion
We previously reported that we have not identified any structural changes in whole brain volumes, cortical thickness and hippocampal subfields with respect to FHD and APOE4 status in this cohort. 36In the present study, we found that individuals with a positive FHD display a non-significant trend towards a smaller medial thalamus, but not the thalamus itself, compared to those without FHD.We did not find any significant differences in cognitive performance on several tests with respect to FHD status alone.Greater age was associated with smaller volumes of all thalamic regions, except the medial thalamus in individuals without FHD.A larger volume of the MDm nucleus was associated with faster processing speeds in those without FHD.Comparatively, the volumes of these nuclei were not associated with cognitive performance in the FHD-positive group.
We did not find any significant volumetric or cognitive differences with respect to APOE4 alone.However, larger volumes of the thalamus and posterior thalamus, specifically the MGN, LSG, PuA, PuL and PuM, were associated with worse performance on the immediate recall test in APOE4 carriers.
To our knowledge, this is the first study to investigate volumetric changes in the thalamic nuclei in a preclinical cohort at midlife.2][43][44] Volumes of several thalamic nuclei such as the mediodorsal, centromedian, pulvinar and medical geniculate are reduced in MCI and Alzheimer's dementia. 25,45ardilla-Delgado et al. 46 did not find any significant volumetric differences in the thalamus or its subregions but did find smaller volumes of the medial thalamus were associated with amyloid and tau pathology in middle-aged PSEN1 carriers, largely made up of individuals who were cognitively unimpaired and a small number with MCI. 46We have identified some non-significant, subtle changes in the thalamic subregions, but it could be that our cohort is too young to observe a significant change yet as it may only be evident in those with higher disease burden.
Greater age was associated with smaller volumes of all thalamic subregions, except for the medial thalamus, in individuals without FHD.It could be that we do not see the same relationship with age in individuals with FHD as the volumes of the thalamus would have already started to decline as part of the disease process. 16,248][49] Thalamic nuclei appear to atrophy at different rates with increasing age in cognitively unimpaired individuals. 50The anteroventral, lateral geniculate, MGN, centromedian and pulvinar are some of the nuclei with higher atrophy rates. 50Interestingly, Pardilla-Delgado et al. 46 reported a significant negative correlation between age and volumes of the thalamus, and the medial and posterior subregions, but not the other subregions, in non-carriers of the PSEN1 mutation, although this study did not perform correction for multiple comparisons and so results should be considered preliminary.Our results are similar to that from published studies, but it is worth noting that we did not find the same relationship between age and the medial thalamus in individuals without FHD.Our results may indicate the medial thalamus is the last region to atrophy in normal aging.Alternatively, there may be subtle changes occurring in that region with increasing age, which the segmentation algorithm is not sensitive enough to identify.We found a larger volume of the MDm was associated with a faster processing speed in individuals without FHD.][53] Functional MRI (fMRI) has shown increased connectivity in healthy individuals between the hippocampus and several thalamic nuclei such as the mediodorsal, laterodorsal and medioventral at the encoding stage to be associated with delayed recall ability of previously learned associations. 54Successful recall tasks have been associated with greater activation of the mediodorsal nucleus and prefrontal cortex during encoding and retrieval, indicating a role for these areas in recall and recognition. 28Bilateral MDm lesions in rhesus monkeys impair the acquisition of new memories while leaving previously learned memories intact. 55Though we did not find an effect of medial thalamic nuclei volumes and cognition in the FHD-positive group, it could be possible that smaller volumes of these regions may affect cognition later in the disease process.
The effect of APOE4 in midlife is not fully understood.While we did not find any significant volumetric differences, Cacciaglia et al. 56 reported a significant increase in the volume of the right medial thalamus in middle-aged, cognitively unimpaired APOE4 carriers, in a dose-dependent manner, compared to non-carriers.8][59] Others have found carriers exhibit a greater decline in memory and cognition over time compared to non-carriers. 5,59,60Li et al. 61 reported APOE4 carriers had reduced hippocampal functional connectivity to areas such as the thalamus, and episodic memory was positively correlated with hippocampal functional connectivity in carriers, highlighting connectivity changes in components of the Papez circuit evident at midlife.
Rather surprisingly, we found larger volumes of several nuclei in the posterior thalamus were associated with worse performance in the immediate recall test in APOE4 carriers.Cacciaglia et al. 58 demonstrated APOE4 mediates opposite relationships between grey matter volumes and episodic memory and executive function.Scarmeas et al. 62 have reported increased perfusion to regions including the insula, pulvinar and cuneus, accompanied by decreased perfusion to parietal and frontal regions in Alzheimer's dementia, and this is negatively correlated with Mini Mental State Examination and recall performance. 62Our findings could indicate larger volumes of the posterior thalamus and its nuclei may not confer benefit on recall ability.Alternatively, it may represent a change in one area that may only occur in conjunction with alterations with other structures that we have not yet identified.van de Mortel et al. 26 have found the thalamus is affected early in the disease course, even prior to hippocampal atrophy, suggesting it may be one of the first signs of cognitive decline.The thalamus may be affected early as it forms part of the Papez circuit, which is important for episodic memory. 10he thalamus aids in several processes in addition to cognition, such as sleep and hearing, which are affected in the prodromal stages of Alzheimer's disease, which also indicates an early involvement of the thalamus in the disease process. 2,63,64There has been little study of the role of thalamic nuclei in Alzheimer's disease, and this may be because the precise function of all nuclei is not yet known.As we learn more about the functions of specific nuclei, we will be able to better understand how the thalamus and its nuclei aid in cognition, hearing and sleep.Greater understanding of this could lead to the development of novel neuroimaging biomarkers.
Strengths of our study include our relatively large sample size, as 611 subjects were included in the final analysis.Furthermore, we used a validated segmentation software to segment the thalamus into its nuclei.These scans then underwent a visual quality control inspection to remove any scans with evidence of incidental pathology or failed segmentation to ensure these results did not affect the analysis.To account for differences in scanners used in our five study sites, we performed ComBat harmonization on the volumetric data.
Limitations of our study include the use of 3 T MRI.Our volumetric results did not survive FDR correction, and as such, these results should be considered preliminary, potentially showing some underlying trends, and require replication in other cohorts.A 7 T MRI may be better to identify any subtle volumetric changes.The cross-sectional nature of this study did not permit investigation of the progression of volumetric changes and the effect on cognition over time.In future studies, additional MRI modalities, such as fMRI, may be used to identify functional connectivity changes in the investigated ROI at this stage.Another limitation is that although sizeable, our study may not have been large enough to detect a difference, especially if it is very subtle.Although we could not find a study with published volumes of thalamic nuclei in preclinical stages in individuals at risk of late-onset Alzheimer's disease, middle-aged PSEN1 mutation carriers have demonstrated a significant difference in the volume of the left thalamus (6962 mm 3 ) in presymptomatic PSEN1 mutation carriers compared to healthy controls (7177 mm 3 ). 42If our population showed similar differences, a much smaller sample size than included here [of 124 in each group (high risk versus low risk)] would have a power of 80%, assuming an alpha value of 0.05 to detect such a difference, though we acknowledge an autosomal dominant group at risk of early-onset Alzheimer's disease may show larger effects than our sporadic dementia group.A final limitation is that we did not investigate the relationship between volumes of the ROI and markers of Alzheimer's disease pathology (Aβ 42 and tau), which are not currently available for the cohort.
In conclusion, we did not find any significant volumetric differences in the thalamic nuclei in relation to FHD or APOE4 allele status, although individuals with a FHD may display a trend towards a smaller volume of the medial thalamus.Greater age was associated with smaller volumes in all thalamic subregions except the medial thalamus in those without FHD.A larger volume of the MDm nucleus was associated with faster processing speeds in individuals without FHD.Contrastingly, larger volumes of several nuclei in the posterior thalamus are associated with worse performance in the immediate recall test in APOE4 carriers.Our findings may indicate an initial dysregulation process, whereby the

Figure 2 The effect of dementia risk on thalamic volumes.
Boxplots showing the differences in the raw, unadjusted volumes of the (A) thalamus (robust linear regression, t = −0.680,

Figure 3 Interaction between age and FHD on thalamic volumes.
Marginal effects plots showing the interaction between age and FHD in predicting changes in the volumes of the (A)